The present invention concerns novel approaches for preparation by synthesis of the 3-phosphate derivatives of 1D-1-(1xe2x80x2,2xe2x80x2-di-O-fattyacyl-sn-glycero-3xe2x80x2-phospho)-myo-inositols (PtdIns), referred to as the D-3-phosphorylated phosphoinositides or the 3-PPI (FIG. 1), their structural and stereochemical analogues, and key starting materials and intermediates of these approaches.
3-PPI are relatively new members of the phosphoinositide group of cellular lipids with emerging critical roles in intracellular signalling. Synthetic 3-PPI and analogues are needed as reagents for defining their biological functions, and for developing diagnostics and therapeutics.
The 3-PPI (FIG. 1) including phosphatidylinositol-3-phosphate, PtdIns(3)P, and the bius- and tris-phosphate derivatives PtdIns(,43)P2 and PtdIns(3,4,5)P3, have been found in eukaryotic cells (1), and the occurrence of PtdIns (3,5)P2 has been suggested (2). These compounds have been demonstrated as activators of protein kinase C isoforms xcex4, xcex5, and n (3), and are putative messengers in cellular signal cascades pertinent to inflammation, cell proliferation, transformation, protein kinesis, and cytoskeletal assembly (4). Minute quantities are found in cells and biochemical studies to determine the cellular targets of the 3-PPI, their metabolic fate, and their roles in the cell cycle have been handicapped because 3-PPI have not been available. Methods for synthesis of 3-PPI have been sought recently (5). These prior art methods suffer from some unique and common problems related respectively to the choice of starting materials for the myo-inositol as well as the diacylglycero-lipid moieties in the 3-PPI. In contrast with the present invention, all start with sn-1,2-diacylglycerols as the lipid moiety in the 3-PPI, and consequently are prone to problems of poor chemical stability endemic to 1,2-diacylglycerols. The latter isomerize readily via neighboring O-acyl migration to equilibrium mixtures comprising the 1,2-, 1,3- and 2,3-diacylglycerols (6). This equilibration is tantamount to racemization which is virtually complete for sn-1,2-di(short-chain)fattyacylglycerols. Therefore, resulting 3-PPI may contain racemic 1,2-2,3- and 1,3-difattyacyl structures, especially with hexanoyl and related short-chain fattyacyls.
Accordingly, it is a principal object of the present invention to provide novel general approaches to synthesis, including novel starting materials, reaction sequences, and novel intermediate compounds, for preparation of the 3-PPI and structural analogues, all of unambiguous structure and absolute stereochemistry in the myo-inositol as well as the sn-glycerol moieties. The present starting materials, reaction sequences, and intermediate compounds, individually and collectively, have utility as materials and processes for obtaining the 3-PPI. The 3-PPI and analogues, in turn, have utility not only as research reagents but also for the development of diagnostics and therapeutics based on the roles of 3-PPI in intracellular signalling. In similar investigations of the biological roles of other bioactive compounds, analogues with reporter groups such as fluorescent tags, are often useful, and so intermediates of 3-PPIs conjugatable to reporter groups are sought.
Broadly, the invention embodies two complementary strategic approaches, and the starting materials and intermediates involved in each, based respectively on (i) synthesis from novel enantiomerically pure myo-inositol derivatives and phosphatidic acids, and (ii) partial synthesis by regioselective-3-phosphorylation of preformed phosphatidylinositol or derived phosphates.
According to one embodiment of the invention, synthesis is carried out by a novel unified approach which is suitable for facile synthesis of all cellular PtdIns-3-phosphates. It is based on the retrosynthetic analysis shown for PtdIns(3,4,5)P3 as an example in FIG. 2. The approach has several novel features. One, it uses 1D-1,2:4,5-di-O-cyclohexylidene-3-O-allyl-myo-inositol (xe2x88x92)-1 as purposely designed starting material and 1D-1,2-O-cyclohexylidene-3-O-allyl-6-O-benzyl-myo-inositol (+)-3 as a key myo-inositol synthon. Two, it incorporates strategic O-protection by a sequentially invariant removal of allyl, 4-methoxybenzyl, and benzyl protecting groups from the inositol hydroxyls destined to appear in the target structures as phosphate, phosphatidyl, and free hydroxyl respectively. Three, it employs preformed 1,2-di-O-fattyacyl-sn-glycero-3-phosphoric acid (sn-3-phosphatidic acid) as the lipid synthon for coupling to appropriately O-protected myo-inositol by a phosphodiester condensation. The sn-3-phosphatidic acid are relatively stable compounds with well established absolute stereochemistry, and their application in the present invention avoids the problems of structural and stereochemical isomerization associated with the application of sn-1,2-fattyacylglycerol in the prior art. As a consequence, the approach uniquely provides unambiguous structural and stereochemical control in the myo-inositol as well as the sn-glycerol moieties, and is applicable for both short-and long-chain fattyacyl types (7). Compared with the long-chain types, the short-chain phosphoinositides are considered to be more useful in biochemical investigations (3, 4). The phosphodiester condensation products are substrates for lipolytic enzyme phospholipase A2 and thus are valuable for incorporating additional useful structural features at a relatively late stage in synthesis. For instance, after lipolysis followed by esterification to introduce (xcfx89-amino-fattyacyls at the sn-glycerol-2 position, the xcfx89-amino group may be conjugated to fluorescent and related reporter groups. The aforementioned attributes are useful and these distinguish the present invention from related literature methods cited above (5).
According to another embodiment of the invention, partial synthesis is based on the retrosynthetic analysis illustrated for PtdIns(3,4,5)P3 from PtdIns(4,5)P2 in FIG. 3. It comprises the regioselective 3-phosphorylation of preformed phosphatidylinositol or derived phosphates but lacking the D-3-phosphate, for the synthesis of the 3-PPI. The preformed PtdIns obtained from natural plant or animal cell sources contain (poly)unsaturated fattyacyls. Using such natural or the corresponding synthetic phosphatidylinositols with unsaturated fattyacyls as the starting materials for 3-phosphorylation as disclosed in the present invention provides methods for the synthesis of 3-PPI containing (poly)unsaturated fattyacyls. These 3-PPI have special physical properties such as lower chain melting transitions for the fattyacyls than for the corresponding saturated fattyacyls, and special bioactivity related to the number, location, and stereochemistry of the double bonds in the fattyacyl chain, and so are desirable. These 3-PPI cannot be prepared by the literature methods (5).